1. Field of the Invention
The present invention relates to a method for producing an enzyme immobilizing carrier using regenerated porous chitosan in particles. The enzyme immobilizing carrier in accordance with the present invention is preferably used as a carrier for immobilizing a variety of enzymes that require carriers having hydrophobic groups.
Furthermore, the present invention relates to an immobilized lipase having excellent catalytic activity in the hydrolysis, synthesis or exchange reaction of ester bonds, where the enzyme is used in an organic solvent. More particularly, the present invention is to provide an immobilized lipase capable of asymmetric synthesis at a higher efficiency than free lipase, in an organic synthesis via the exchange reaction of a variety of esters.
2. Prior Art
It is known that cross-linked products of regenerated porous chitosan in particles are used extensively as enzyme immobilizing carriers.
The advantages of regenerated porous chitosan in particles include that the material is from a natural origin so it is quite safe; that larger pores are more uniformly present from the surface to the inside of the carrier when chitosan is used as a carrier than synthetic resin carriers, thus providing greater substrate diffusibility; that highly reactive amino groups convenient for immobilizing enzymes via covalent bonding are disposed within the molecule; and that chitosan itself has a higher affinity with the enzyme so that a higher amount of the enzyme may be immobilized onto chitosan.
Carriers of regenerated and cross-linked chitosan in particles, which is regenerated polysaccharide, have advantages, in that the stability thereof in an organic solvent is very high, in addition to the hydrophilicity and porosity of the products. However, the products have drawbacks, in view of their potency to express enzyme activity because the carriers of regenerated porous chitosan that is cross-linked with polyfunctional reagents are highly hydrophilic. Such carriers, when used for proteases and enzymes catalyzing carbohydrates and for the group of enzymes whose substrates are hydrophobic substances like lipid, have reduced expressed enzyme activity, since proteases and enzymes catalyzing carbohydrates require a certain degree of hydrophobicity for the carrier.
Japanese Patent Publication No. Sho 63-54285 discloses that porous chitosan in particles that is cross-linked with 4,4'-diphenylmethane diisocyanate or hexamethylene diisocyanate may be used to immobilize enzyme groups, such as proteases and enzymes catalyzing carbohydrates, that require a certain degree of hydrophobicity for the immobilizing carrier, and the group of enzymes whose substrates are hydrophobic substances such as lipids. However, the carriers have lower immobilized enzyme activity and lower expressed enzyme activity.
Recently, active research works has focused on the application of chitosan to enzymatic organic synthesis. This is due to the excellent characteristic properties of enzymes, wherein enzymes are reactive at ambient temperature and ambient pressure such that thermally unstable substances can be synthesized via enzymes, enzymatic reactions progress in an energy-saving manner without causing pollution, and enzymes have good reaction specificity, such as position specificity, substrate specificity and capability of asymmetric synthesis.
Enzyme reactions in organic solvents, in particular, are drawing attention, from the respect that hydrolases can be applied to various synthesis and transition reactions. Compared with other enzymes, many lipid hydrolases, such as lipases are so highly resistant to organic solvents that such lipases can facilitate reactions for ester exchange and ester synthesis at a high efficiency. Thus, extensive research work has been conducted. Because the production and purification of lipases demand laborious work, however, the development of an immobilized lipase of a higher efficiency has been an issue of importance for the industrial application of lipase.
A review of lipase immobilization is found in "Journal of American Oil Chemist's Society", Vol.67, pp.890-910 (1990), where examples of representative lipase immobilizing carriers are illustrated, including inorganic carriers such as diamatoceous earth, silica, porous glass, etc.; various synthetic resins and synthetic resin ion exchangers; and natural polysaccharide carriers such as cellulose and cross-linked dextrin introduced with ion exchange groups. According to this reference, it is reported that these carriers are grouped as either hydrophilic carriers and hydrophobic carriers, and that lipase immobilized on a hydrophobic carrier such as synthetic resin expresses a higher ester exchange activity than lipase immobilized on a hydrophilic carrier.
An example of a lipase immobilized onto a synthetic resin ion exchanger is disclosed in Japanese Patent Laid-open No. Hei 4-287689. According to this reference, lipase from Pseudomonas immobilized on Amberlite XAD-2 exerts an activity for the ester exchange reaction between acetate vinyl monomer and .alpha.-D,L-phenylethyl alcohol. Although such synthetic resin ion exchanger has higher hydrophobicity as a carrier, the exchanger has drawbacks such as solubilization of the residual monomers and swelling of the resin when the immobilized lipase is used for an enzyme reaction in an organic solvent, although such use is important for an immobilized lipase.
As a method for immobilizing lipase, alternatively, the treatment of lipase with phospholipid or fatty acid is reported. For immobilizing lipase, methods for treating an immobilizing carrier with phospholipid or fatty acid are disclosed in Japanese Patent Laid-open No. Sho 62-134090, Japanese Patent Laid-open No. Hei 1-153090, and Japanese Patent Laid-open No. Hei 4-335893. According to any of the methods disclosed therein, phospholipid or fatty acid immobilized onto an immobilizing carrier via adsorption or hydrophobic bonding is desorbed from the carrier during the use of the carrier, leading to a lower efficiency.